Effect of bone marrow mesenchymal stem cell on the expression of IL-10 and TNF-α in mice with ischemia-reperfusion acute kidney injury / 器官移植

Objective To evaluate the effect of bone marrow mesenchymal stem cell (BMSC) on the expression of interleukin (IL)-10 and tumor necrosis factor (TNF)-α in mice with ischemia-reperfusion acute kidney injury (IR-AKI). Methods All mice were randomly divided into the sham operation group (control group), ischemia-reperfusion injury group (IRI group) and BMSC treatment group (BMSC group), with 6 mice in each group, respectively. The renal function and pathological changes of mice were detected. The cell apoptosis of renal tissues of mice was determined. The expression levels of serum IL-10 and TNF-α of mice were quantitatively measured. The mouse BMSC was randomly divided into the control and hypoxia-reoxygenation groups (IRI group), and the expression levels of IL-10 and TNF-α in cell supernatant were determined. Results The renal structure of mice was normal in the control group, severe damage was observed in the IRI group, and mild damage occurred in the BMSC group. Compared with the control group, the renal tissue injury scores were significantly higher in the IRI and BMSC groups (both P < 0.05). Compared with the IRI group, the renal tissue injury score was significantly lower in the BMSC group (P < 0.05). Compared with the control group, the levels of serum creatinine (Scr) and blood urea nitrogen (BUN) were remarkably up-regulated in the IRI group, and the level of BUN was significantly up-regulated in the BMSC group (all P < 0.05). Compared with the IRI group, the levels of Scr and BUN were significantly down-regulated in the BMSC group (both P < 0.05). In the IRI group, the quantity of apoptotic cells in the renal tissues was considerably higher than those in the BMSC and control groups, and the quantity of apoptotic cells in the BMSC group was significantly higher than that in the control group (all P < 0.05). Compared with the control group, the levels of serum IL-10 and TNF-α were significantly up-regulated in the IRI group, whereas the level of serum TNF-α was significantly down-regulated and the level of serum IL-10 was significantly up-regulated in the BMSC group (all P < 0.05). Compared with the IRI group, the levels of serum IL-10 and TNF-α were significantly down-regulated in the BMSC group (both P < 0.05). The levels of IL-10 and TNF-α in the cell supernatant did not significantly differ between the IRI and control groups (P=0.080、0.627). Conclusions BMSC infusion may reduce the incidence of renal IRI and inflammation, probably via the mechanism of down-regulating TNF-α expression rather than up-regulating IL-10 expression.

The application value of Multi-Latex polygranular technique joint detection of urinary microproteins in noninvasive diagnosis after renal transplantation / 器官移植

Objective To investigate the application value of Multi-Latex polygranular technique joint detection of kidney injury-related urinary microproteins in noninvasive diagnosis after renal transplantation. Methods Clinical data of 72 recipients undergoing renal transplantation were retrospectively analyzed. According to the level of serum creatinine (Scr), the recipients were divided into normal renal function group (group A, n=14), mild kidney injury (group B, n=37), and severe kidney injury group (group C, n=21). 20 healthy volunteers were selected as the healthy control group (HC group). The contents of urinary retinol binding protein (RBP), microalbumin (mAlb), IgG, transferrin (TRF), α1-microglobulin (MG), and β2-MG of subjects in each group were detected using the Multi-Latex polygranular technique. The correlation between urinary microproteins and Scr, blood urea nitrogen (BUN) was analyzed. The differences of urinary microproteins in each group were compared. And the diagnostic value of single and joint detection of urinary microproteins was evaluated. Results Six kinds of urinary microproteins in HC group and group A were significantly lower than those in group B and group C, and six kinds of urinary microproteins in group B were significantly lower than those in group C (all P < 0.01). Six kinds of urinary microproteins in renal transplant recipients were positively correlated with BUN. RBP, mAlb, α1-MG, and β2-MG were positively correlated with Scr. The correlations were statistically significant (P < 0.001-0.05). The diagnostic value of joint detection of urinary microproteins is better than the detection of single index, among which TRF+mAlb+RBP+α1-MG quadruple detection had the highest diagnostic value. Conclusions Six kinds of urinary microproteins can be used as specific indicators to reflect graft renal function. The polygranular technique can simultaneously detect its contents and achieve noninvasive diagnosis. The diagnosis based on TRF+mAlb+RBP+α1-MG quadruple detection is expected to further improve the noninvasive diagnosis system after renal transplantation.

Dynamic changes of lymphocyte subsets and their correlation with renal function in recipients with stable graft status after renal transplantation / 器官移植

Objective To investigate the dynamic changes of peripheral blood lymphocyte subsets and their correlation with renal function in recipients with stable graft status after renal transplantation. Methods Forty-five recipients who underwent renal transplantation for the first time and had stable graft function within postoperative 6 months were selected. The proportion and absolute value of lymphocyte subsets were detected by flow cytometry (FCM) in 180 peripheral blood samples from recipients at 15 d, 1, 3 and 6 months after renal transplantation. The dynamic changes of lymphocyte subsets with the extension of postoperative time and their correlation with serum creatinine (Scr) and blood urea nitrogen (BUN) were analyzed. Results The Scr levels did not significantly differ at 4 time points after renal transplantation (all P > 0.05). The BUN levels significantly differed between 15 d and 1 month after renal transplantation, and between 1 and 3 months after renal transplantation (P=0.002, P=0.001). The proportion of CD3+CD8+T cells, CD3+CD4+T cells, natural killer (NK) cells and CD4/CD8 ratio at postoperative 15 d significantly differed from those at 1 month after operation (P=0.009, P=0.004, P < 0.001, P=0.004). The proportion of B cells significantly differed between 15 d and 1 month, and between 1 and 3 months after renal transplantation (both P < 0.001). The absolute values of CD3+T cells, CD3+CD8+T cells, CD3+CD4+T cells and NK cells at postoperative 15 d significantly differed from those at 1 month after renal transplantation (P=0.001, P=0.002, P=0.003, P < 0.001). The absolute values of CD3+CD8+T cells significantly differed between 3 and 6 months after operation (P=0.015). The absolute value of B cells at 1 month after renal transplantation significantly differed from that at 3 months after renal transplantation (P=0.001). The proportion and absolute value of lymphocyte subsets were not significantly correlated with the Scr level (both P > 0.05). The proportion and absolute value of CD3+CD8+T cells and NK cells were negatively correlated with BUN (P < 0.001-0.05), whereas the proportion of CD3+CD4+T cells and B cells was positively correlated with the BUN level (P < 0.001-0.05). The absolute value of CD3+T cells was negatively associated with the BUN level (P < 0.05). Conclusions T cells and NK cells in the lymphocyte subsets of stable recipients raise to the stable state within 1 month after renal transplantation, whereas B cells decrease to stable state within 3 months renal transplantation. The dynamic changes of lymphocyte subsets are correlated with the BUN level.

Brucellosis after renal transplantation: a report of one case and literature review / 器官移植

Objective To analyze and summarize the clinical features and diagnosis and treatment experience of brucellosis after renal transplantation. Methods Clinical data of one case with brucellosis after renal transplantation admitted to the 309thHospital of Chinese People's Liberation Army in October 2016 was collected. The clinical features, diagnosis and treatment were retrospectively analyzed. Clinical experience was summarized and literature review was conducted. Results At 3 months after renal transplantation, the patient suffered from temperature rise without known causes and presented with fever in the morning with a duration of 3 d. The route of infection was unknown, and the symptoms of alternative types of infection were not obvious. Empirical anti-infectious therapy was delivered for 1 week and yielded no efficacy. Blood culture test confirmed the diagnosis of brucella melitensis infection. The treatment included anti-infecting by the rifampicin, doxycycline, sulfamethoxazole, preventing the incidence of complications actively and protecting the liver and renal function. High clinical efficacy was achieved. During the 1-year follow up after discharge, the renal graft was stable and no other infectious symptoms, such as fever was found. Conclusions Brucellosis with unknown route of infection after renal transplantation is extremely rare and the common symptom is Malta fever. When the empirical anti-infectious treatment is not effective, blood culture and other related tests should be performed to confirm the diagnosis. The combination of rifampicin and doxycycline is recommended.

Diagnostic value of flow cytometry in postoperative infection after renal transplantation / 器官移植

Objective To assess the value of flow cytometry in the diagnosis of postoperative infection following renal transplantation. Methods According to postoperative imaging findings and laboratory examination outcomes, 51 recipients undergoing the first renal transplantation were divided into the bacteria (n=33), fungus (n=9) and BK virus (n=9) groups. Twenty-eight recipients with stable conditions after renal transplantation were assigned into the stable group. Flow cytometry was adopted to detect the percentage and absolute counting of lymphocyte subpopulation in the peripheral blood of recipients in each group. Renal function, percentage and absolute counting of lymphocyte subpopulation in the peripheral blood were statistically compared among different groups. Receiver operating characteristic (ROC) curve was drawn to evaluate the diagnostic value of the percentage and absolute counting of lymphocyte subpopulation in infectious diseases after renal transplantation. Results Compared with the stable group, the serum creatinine (Scr) and blood urea nitrogen (BUN) levels in the bacteria, fungus and BK virus groups were significantly up-regulated, respectively (P=0.035, 0.007, 0.024; 0.037, 0.006, 0.032). Compared with the stable group, the percentage of CD16+CD56+natural killer (NK) cells was significantly declined in the bacterial (P=0.036) and fungus groups (P=0.015), and the proportion of CD4+/CD8+T cells was dramatically decreased in the fungus group (P=0.004). Compared with the bacterial group, the percentage of CD3+CD8+T cells was significantly elevated (P=0.013 and 0.008), the proportion of CD3+CD4+T cells was considerably declined (P=0.003 and 0.010), and the percentage of CD4+/CD8+T cells was significantly declined (P=0.003 and 0.005) in the fungus and BK virus groups. Compared with the stable group, the quantity of CD3+T cells, CD3+CD8+T cells and CD16+CD56+NK cells was significantly declined in the bacterial, fungus and BK virus groups, respectively (P=0.025, 0.002, 0.003; 0.015, 0.005, 0.006; 0.001, 0.001, 0.031). In addition, the quantity of CD3+CD4+T cells was considerably decreased in the fungus and BK virus groups (P=0.001, 0.003). The quantity of CD19+B cells was significantly reduced in the BK virus group (P=0.019). Compared with the bacterial group, the quantity of CD3+CD4+T cells was considerably lower in the fungus group (P=0.023). ROC curve analysis revealed that the quantity of CD3+CD4+T cells [area under curve(AUC)=0.8492] and CD16+CD56+NK cells (AUC=0.8889) yielded relatively high accuracy in the diagnosis of fungal infection. The quantity of CD3+T cells (AUC=0.8472), CD3+CD4+T cells (AUC=0.8452) and CD19+B cells (AUC=0.8115) yielded relatively high accuracy in the diagnosis of BK virus infection. Conclusions Flow cytometry detection of the lymphocyte subpopulation in peripheral blood can evaluate the immune function of patients. Absolute counting of lymphocyte subpopulation can directly assess the degree of immunity. These two combined parameters provide guiding significance for the diagnosis and differential diagnosis of infectious diseases in recipients after renal transplantation.

Activation of CD8+T cells regulated by γ-aminobutyric acid and its receptors / 器官移植

Objective To evaluate the effect of γ-aminobutyric acid (GABA) and its receptors upon the proliferation of CD8+T cells.Methods The splenic CD8+T cells of Balb/c mice were obtained by CD8+f cell magnetic bead sorting kit.Under the effect of CD3/CD28-activated magnetic bead,CD8+T cells were stimulated by different concentrations of GABA.5-bromo-2-deoxyuridine (BrdU) labeling and flow cytometry were performed to detect the proliferation of CD8+T cells.The expression levels of GABA-A and GABA-B receptor before and after CD8+T cell activation were compared by fluorescent quantitative real-time polymerase chain reaction (PCR).Result Flow cytometry result revealed that GABA could inhibit the proliferation of activated CD8+T cells,manifested as significant decrease in the quantity of CD152+CD8+T cells.Fluorescent quantitative real-time PCR demonstrated that GABA-A receptor subtypes α2,α6 and GABA-B receptor subtype 1a were expressed only when the CD8+T cells were activated.After CD8+T cell activation,the quantity of GABA-A receptor subtypes α3,αs,β2,β3,γ1,γ2 and θ were significantly increased,whereas the quantity of GABA-B2R and GABA-B1b did not significantly differ before and after CD8+T cell activation.Conclusions GABA can suppress the proliferation of activated CD8+T cells.The activation of CD8+T cells is regulated by GABA receptors.However,the underlying mechanism remains to be elucidated.

Changes of erythrocytes surface molecule CD35,CD58 and CD59 expression in recipients infected with cytomegalovirus after renal transplantation / 器官移植

Objective To investigate the change rules and its significance of erythrocytes surface molecule CD35 , CD58 and CD59 expression in recipients infected with cytomegalovirus (CMV)after renal transplantation. Methods Eighty-two recipients undergoing allogeneic renal transplantation were selected and divided into the negative (n=21 )and positive CMV groups (n=61 )based on the qualitative detection of CMV-pp65 antigen in peripheral blood. According to the results of CMV-pp65 (+)leucocyte count,all 61 patients in positive CMV group were further divided into low (n=55)and high active infection subgroups (n =6 ). Healthy adults were recruited into the normal control group (n =30 ). The expression levels of CMV-pp65 antigen,erythrocytes surface molecule CD35,CD58 and CD59 were measured by flow cytometry. Results Compared with normal control group,the expression levels of erythrocytes surface molecule CD35 , CD58 and CD59 in the positive CMV group were significantly down-regulated,and the CD35 and CD59 expression in the negative CMV group were considerably down-regulated (all P<0. 05 ). Compared with negative CMV group,the expression levels of CD58 and CD59 in the positive CMV group were significantly down-regulated (both P<0. 05 ). The expression levels of CD35 and CD59 in the high active infection subgroup were significantly lower than those in the low active infection subgroup (both P<0. 05 ). Conclusions The more severe active CMV infection after renal transplantation,the lower expression of erythrocytes surface molecule CD35,CD58 and CD59,hinting that red cell immune dysfunction is probably involved with active CMV infection.

Isolation, culture and surface markers detection of human umbilical cord mesenchymal stem cells / 白血病·淋巴瘤

Objective To investigate the methods of isolation and culture in vitro and detect the surface markers of human umbilical cord mesenchymal stem cells.Methods Human umbilical cord Wharton' s jelly was separated and cut up as small as possible,and then cultured with α-MEM.Human umbilical cord mesenchymal stem cells could be obtained by culturing the tissue block adhered the bottle wall.And the cells were passaged at a certain density.The surface markers of human umbilical cord mesenchymal stem cells were detected by FACS when the cells were in Generation Three.Results Human umbilical cord mesenchymal stem cells were obtained from Wharton' s jelly conveniently,with fibroblast shape and stable proliferation and passage.CD29,CD44,CD105 were strongly expressed on human umbilical cord mesenchymal stem cells.But CD45,CD34,HLA-DR,HLA-G,CD80,CDs6 were not expressed.Conclusion Human umbilical cord mesenchymal stem cells can be obtained effectively from the culture of the tissue block,which provides a rich source of cells for tissue engineering.

γ-aminobutyric acid B receptor (GABABR)ameliorated liver fibrosis by inhibiting hepatic cell migration / 中华肝胆外科杂志

Objective To investigate the role of r-aminobutyric acid B receptor in the development of liver fibrosis.Methods Thirty-two Sprague-Dawley (SD) rats were divided into four groups including a control group,a model group,a baclofen group,and a CGP35348 group.Liver fibrosis was then induced by carbon tetrachloride (CCl4).Baclofen and CGP35348 treatment were carried out after the formation of liver fibrosis,followed by complete extraction of the eyeball to obtain blood sample to test liver function.Liver tissue specimens were cut and stored for histological staining,histochemistry,real-time polymerase chain-reaction (RT-PCR),and western blot analysis.Results Histological staining indicated that the degree of liver fibrosis was more severe in the CGP35348 group than in the baclofen group (P＜0.001).The levels of alanine transaminase (ALT),aspartate aminotransferase (AST),gamma-glutamyl transferase (GGT),total bilirubin (TBil),and direct bilirubin (DBil) were significantly lower in the baclofen group than in the CGP35348 group (P＜0.01).The levels of ALT,AST,GGT,TBil,and DBil were significantly higher in the CGP35348 group than in the model group (P＜0.05).Immunofluorescence results show that the hepatic cell migration was inhibited in the baclofen group.Western blot results showed that the expression levels of α-SMA protein were significantly lowered in the baclofen group when compared to that of the CGP35348 group and model group (P＜0.01).Conclusion GABAB receptor might play a role in the liver protection by inhibition of migration of hepatic cells in liver fibrosis.Further studies into the mechanism behind this function are further needed and may be a potential source of future anti-fibrotic treatment.

Impacts of concentrations of Tacrolimus on NK cells and receptors in peripheral blood of renal transplant recipients / 中华器官移植杂志

Objective To study the effects of tacrolimus(Tac) concentrations on the number of NK cells and receptor expression in peripheral blood of renal transplantation receptors.Methods A total of 60 first-time kidney transplantation recipients in our institute from Dec.2007 to July 2009 were followed up.Tac maintenance immunosuppressive therapy was given to all recipients.The recipients were divided into low-concentration Tac group (6.84 + 1.72μg/L,n =30) and highconcentration Tac group ( 11.88 + 2.59 μg/L,n =30) according to concentrations of Tac.Twenty healthy volunteers served as controls.Before and 6 months after operation,concentrations of Tac were analyzed by using micro particle immunoassay chemiluminescent method.NK cells and their receptors (CD85j,CD158d,CD94 and NKG2D) were detected by using flow cytometry.The concentrations of soluble HLA-G5 were detected by using ELISA.Results The number of NK cells in lowconcentration Tac group and high-concentration of Tac group preoperatively was significantly reduced as compared with control group (P ＜ 0.05 ). The percentage and number of NK cells in low concentration Tac group and high-concentration Tac group at 6th month after operation were significantly reduced as compared with control group (P＜0.05).The number of NK cells in lowconcentration Tac group was significantly greater than in high-concentration Tac group (P＜ 0.05).There was no significant differende in the expression of CD85j,CD158,CD94 and NKG2D before operation between two groups(P＞0.05).The expression of CD85j and CD158d in two groups was increased,but that of CD94 and NKG2D was decreased at 6th month post-transplantation as comapred with that preoperation.In low-concentration Tac group,the expression of CD85j and CD158d was increased as compared with that in high-concentration Tac group (P＜0.05 ).Spearman correlation analysis revealed that the CD85j and CD158d expression had a positive correlation with sHLA-G5(P＜0.01 ),but the NKG2D had a negative correlation with sHLA-G5(P＜0.01 ).Conclusion There was correlation between the concentrations of Tac and NK cells count and NK receptors. Low concentrations of Tac can safely and effectively protect kidney function.The number of NK cells andtheir inhibitor receptors are increased in the recipients with low concentration of Tac.